Diagnostic assays are widespread and central for the diagnosis, treatment and management of many diseases. Different types of diagnostic assays have been developed over the years in order to simplify the detection of various analytes in clinical samples such as blood, serum, plasma, urine, saliva, tissue biopsies, stool, sputum, skin or throat swabs and tissue samples or processed tissue samples. These assays are frequently expected to give a fast and reliable result, while being easy to use and inexpensive to manufacture. Understandably it is difficult to meet all these requirements in one and the same assay. In practice, many assays are limited by their speed. Another important parameter is sensitivity. Recent developments in assay technology have led to increasingly more sensitive tests that allow detection of an analyte in trace quantities as well the detection of disease indicators in a sample at the earliest time possible.
A common type of disposable assay device includes a zone or area for receiving the liquid sample, a reagent zone also known as a conjugate zone, and a reaction zone also known as a detection zone. These assay devices are commonly known as lateral flow test strips. They employ a porous material, e.g., nitrocellulose, defining a path for fluid flow capable of supporting capillary flow. Examples include those shown in U.S. Pat. Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660 all of which are incorporated herein by reference in their entireties.
The sample-addition zone frequently consists of a more porous material, capable of absorbing the sample, and, when separation of blood cells is desired, also effective to trap the red blood cells. Examples of such materials are fibrous materials, such as paper, fleece, gel or tissue, comprising, e.g., cellulose, wool, glass fiber, asbestos, synthetic fibers, polymers, or mixtures of the same.
Another type of assay device is a non-porous assay having projections to induce capillary flow. Examples of such assay devices include the open lateral flow device as disclosed in WO 2003/103835, WO 2005/089082, WO 2005/118139, and WO 2006/137785, all of which are incorporated herein by reference in their entireties.
A known non-porous assay device is shown in FIG. 1. The assay device 1, has at least one sample addition zone 2, a reagent zone 3, at least one detection zone 4, and at least one wicking zone 5. The zones form a flow path by which sample flows from the sample addition zone to the wicking zone. Also included are capture elements, such as antibodies, in the detection zone 4, capable of binding to the analyte, optionally deposited on the device (such as by coating); and a labeled conjugate material also capable of participating in reactions that will enable determination of the concentration of the analyte, deposited on the device in the reagent zone, wherein the labeled conjugate material carries a label for detection in the detection zone. The conjugate material is dissolved as the sample flows through the reagent zone forming a conjugate plume of dissolved labeled conjugate material and sample that flows downstream to the detection zone. As the conjugate plume flows into the detection zone, the conjugated material will be captured by the capture elements such as via a complex of conjugated material and analyte (as in a “sandwich” assay) or directly (as in a “competitive” assay. Unbound dissolved conjugate material will be swept past the detection zone into the at least one wicking zone 5. Also shown in FIG. 1 are projections or micropillars 7. An instrument such as that disclosed in US 20060289787A1, US20070231883A1, U.S. Pat. Nos. 7,416,700 and 6,139,800 all incorporated by reference in their entireties, are able to detect the bound conjugated material in the detection zone. Common labels include fluorescent dyes that can be detected by instruments which excite the fluorescent dyes and incorporate a detector capable of detecting the fluorescent dyes.
In order to produce a reportable result from a measured signal, e.g., a fluorescent signal, a calibration curve needs to be formulated to correlate the measured signal to the concentration of the analyte of interest in the sample being analyzed. Developing a calibration curve is well known in the art and does not need detailed explanation. Briefly, multiple samples having known varying concentrations of analyte (also called calibrator fluids) are run on an assay device in a manner similar to an end user performing an assay on a sample having an unknown concentration of analyte. The signal produced by the analyte signal producing complex, such as an analyte/labeled antibody conjugate, is read and recorded as a data point for each of the multiple samples. The data points are plotted on a curve of concentration versus signal strength. The data points can then be curve fit into an equation that provides analyte concentration as a function of signal strength for that particular assay. For example, a linear correlation can be represented by C=mS+b, where C is the concentration of the analyte, S is the measured signal and m and b are experimentally determined constants. Non-linear correlations can be represented with non-linear mathematical models such as the logit/log 4 relationship.
For many commercially available assays, the calibration curve is developed at the factory where the assay is made. Due to variation in raw materials and other factors when an assay is made, a different lot-specific calibration curve may be developed for each lot of assays produced. The calibration curve data can then be included in each lot of assay sold to an end user. Alternatively, a calibration curve is automatically created by the customer's analyzer when the customer runs a calibration process with the lot of assay material, their analyzer, and a series of calibrator materials provided by the manufacturer.
When making a calibration curve at a factory, a standard calibrator fluid is used to approximate the characteristics of the actual samples that will be used by the end user of the assay device. For example, if the assay will be used with plasma samples, the calibrator fluid will generally be formulated to mimic the characteristics of a typical plasma sample. This should result in the unknown concentration of the analyte in the sample being assayed being the same as the concentration of the analyte in the calibrator fluid for equivalent measured signals.
A typical measured signal for lateral flow assays is a peak height or peak area of a trace of fluorescent intensity vs. distance along the detection zone. However, it is sometimes the case where the measured signal in sample does not depend on concentration alone. Other factors, particularly for capillary driven lateral flow assays devices, in addition to analyte concentration, can affect the measured signal. If these factors are not taken into account, then the measured signal for a sample being assayed will not accurately correlate to the true concentration of analyte in the sample. This of course, can have profound effects, e.g., on a patient's diagnosis or prognosis.
Accordingly, there is a need for a method that can take into account other factors that affect the measured signal of a capillary driven lateral flow assay device.